The present invention relates to compositions and methods of preparing amino acid chelates that are electrically neutral and essentially free of interfering ions. The composition is prepared by reacting, in an aqueous solution, a calcium oxide and/or hydroxide, an amino acid, and a soluble metal sulfate salt at a ratio sufficient to allow substantially all of the ions present in solution to react forming a metal amino acid chelate, an essentially inert calcium sulfate salt, and water. The metal amino acid chelates of the present invention have a ligand to metal molar ratio from about 2:1 to 3:1.
Amino acid chelates are generally produced by the reaction between xcex1-amino acids and metal ions having a valence of two or more to form a ring structure. In such a reaction, the positive electrical charge of the metal ion is neutralized by the electrons available through the carboxylate or free amino groups of the xcex1-amino acid.
Traditionally, the term xe2x80x9cchelatexe2x80x9d has been loosely defined as a combination of a metallic ion bonded to one or more ligands forming heterocyclic ring structures. Under this definition, chelate formation through neutralization of the positive charges of the divalent metal ions may be through the formation of ionic, covalent, or coordinate covalent bonding. An alternative and more modern definition of the term xe2x80x9cchelatexe2x80x9d requires that the metal ion be bonded to the ligand solely by coordinate covalent bonds forming a heterocyclic ring. In either case, both definitions describe a metal ion and a ligand forming a heterocyclic ring.
A chelate is a definite structure resulting from precise requirement of synthesis. Proper conditions must be present for chelation to take place, including proper mole ratios of ligands to metal ions, pH, and solubility of reactants. For chelation to occur, all components are (generally dissolved in solution and are either ionized or of appropriate electronic configuration in order for coordinate covalent bonding and/or ionic bonding between the ligand and the metal ion to occur.
Chelation can be confirmed and differentiated from mixtures of components by infrared spectra through comparison of the stretching of bonds or shifting of absorption caused by bond formation. As applied in the field of mineral nutrition, there are two allegedly xe2x80x9cchelatedxe2x80x9d products which are commercially utilized. The first is referred to as a xe2x80x9cmetal proteinate.xe2x80x9d The
American Association of Feed Control officials (AAFCO) has defined a xe2x80x9cmetal proteinatexe2x80x9d as the product resulting from the chelation of a soluble salt with amino acids and/or partially hydrolyzed protein. Such products are referred to as the specific metal proteinate, e.g., copper proteinate, zinc proteinate, etc. Sometimes, metal proteinates are even referred to as amino acid chelates, though this characterization is not accurate. This is because by definition, a metal proteinate must contain partially hydrolyzed proteins which may or may not be mixed with amino acids.
The second product, referred to as an xe2x80x9camino acid chelate,xe2x80x9d when properly formed, is a stable product having one or more five-membered rings formed by a reaction between the carboxyl oxygen, and the xcex1-amino group of an xcex1-amino acid with the metal ion. Such a five-membered ring is defined by the metal atom, the carboxyl oxygen, the carbonyl carbon, the xcex1-carbon and the xcex1-amino nitrogen. The actual structure will depend upon the ligand to metal mole ratio and whether the carboxyl oxygen forms a coordinate covalent bond or an ionic bond with the metal ion. Generally, the ligand to metal molar ratio is at least 1:1 and is preferably 2:1 or 3:1. However, in certain instances, the ratio may be 4:1. Most typically, an amino acid chelate may be represented at a ligand to metal molar ratio of 2:1 according to Formula 1 as follows: 
In the above formula, the dashed lines represent coordinate covalent bonds, covalent bonds, or ionic bonds. The solid lines between the xcex1-amino group and the metal (M) are covalent or coordinate covalent bonds. Further, when R is H, the amino acid is glycine which is the simplest of the xcex1-amino acids. However, R could be a radical forming any other of the other twenty or so naturally occurring amino acids derived from proteins. All of the amino acids have the same configuration for the positioning of the carboxyl oxygen and the xcex1-amino nitrogen with respect to the metal ion. In other words, the chelate ring is defined by the same atoms in each instance, even though the R group may vary.
The American Association of Feed Control Officials (AAFCO) have also issued a definition for amino acid chelates. It is officially defined as the product resulting from the reaction of a metal ion from a soluble metal salt with amino acids having a mole ratio of one mole of metal to one to three (preferably two) moles of amino acids to form coordinate covalent bonds. The average weight of the hydrolyzed amino acids must be approximately 150 and the resulting molecular weight of the chelate must not exceed 800. The products are identified by the specific metal forming the chelate, e.g., iron amino acid chelate, copper amino acid chelate, etc. The reason a metal atom can accept bonds over and above the oxidation state of the metal is due to the nature of chelation.
For example, at the xcex1-amino group of an amino acid, the nitrogen contributes both of the electrons used in the bonding. These electrons fill available spaces in the d-orbitals forming a coordinate covalent bond. Thus, a metal ion with a normal valency of +2 can be bonded by four bonds when fully chelated.
In this state, the chelate is completely satisfied by the bonding electrons and the charge on the metal atom (as well as on the overall molecule) is zero. As stated previously, it is possible that the metal ion be bonded to the carboxyl oxygen by either coordinate covalent bonds or ionic bonds. However, the metal ion is typically bonded to the xcex1-amino group by coordinate covalent bonds only.
Amino acid chelates can also be formed using small peptide ligands instead of single amino acids. These will usually be in the form of dipeptides, tripeptides, and sometimes tetrapeptides because larger ligands have molecular weights that are too great for direct cellular assimilation of the chelate formed.
Generally, peptide ligands will be derived by the hydrolysis of protein. However, peptides prepared by conventional synthetic techniques or genetic engineering can also be used. When a ligand is a di- or tripeptide, a radical of the formula [C(O)CHRNH]e H will replace one of the hydrogens attached to the nitrogen atom in Formula 1. R, as defined in Formula 1, can be H or the residue of any other naturally occurring amino acid and e can be an integer of 1, 2 or 3. When e is 1 the ligand will be a dipeptide, when e is 2 the ligand will be a tripeptide and so forth.
The structure, chemistry and bioavailability of amino acid chelates is well documented in the literature, e.g. Ashmead et al., Chelated Mineral Nutrition, (1982), Chas. C. Thomas Publishers, Springfield, Ill.; Ashmead et al., Intestinal Absorption of Metal Ions, (1985), Chas. C. Thomas Publishers, Springfield, Ill.; Ashmead et al., Foliar Feeding of Plants with Amino Acid Chelates, (1986), Noyes Publications, Park Ridge, N. J.; U.S. Pat. Nos. 4,020,158; 4,167,564; 4,216,143; 4,7216,44; 4,599,152; 4,774,089; 4,830,716; 4,863,898; 4,725,427; and others, the entire teachings of which are incorporated by reference.
One advantage of amino acid chelates in the field of mineral nutrition is attributed to the fact that these chelates are readily absorbed in the absorptive mucosal cells or plant cells by means of active transport or other know mechanisms. In other words, the minerals are absorbed along with the amino acids as a single unit utilizing the amino acids as carrier molecules. Therefore, the problems associated with the competition of ions for active sites and the suppression of specific nutritive mineral elements by others are avoided. This is especially true for compounds such as iron sulfates that must be delivered in relatively large quantities in order for the body or plant to absorb an appropriate amount. This is significant because large quantities often cause nausea and other gastrointestinal discomforts in animals as well as create an undesirable taste. Additionally, in plants, large amounts of these compounds can act to burn leaves and cause other undesirable results.
In the past, amino acid chelates have generally been made by first dissolving a water soluble metal salt in water. An amino acid ligand is then reacted with the metal ion at a ratio of ligand to metal from 1:1 to 4:1, preferably 2:1. Often, the ligand is a hydrolysis product obtained by acid, base, base-acid, base-acid-base, or enzyme hydrolysis. In such cases, the by products from hydrolysis, such as anions including chlorides, sulfates, phosphates and nitrates, and cations including potassium and sodium, remain in the hydrolysate. Reaction products of metal salts with proteins or with acid and/or base hydrolyzed proteins are taught in U.S. Pat. Nos. 2,960,406; 3,396,104; 3,463,858; 3,775,132; 4,020,158; 4,103,003; and 4,172,072.
In fact, most water soluble salts used in making amino acid chelates have been either sulfates or chlorides. Using the sulfate ion as exemplary, the reaction has generally proceeded 
where M is a bivalent metal cation and R is a radical of a naturally occurring amino acid, dipeptide or polypeptide. It is apparent from the above formula that the sulfate anion is present in the reaction mixture in the form of sodium sulfate.
U.S. Pat. No. 2,877,253 teaches a product formed by the reaction of one mole of glycine with one mole of ferrous sulfate. That patent indicates that the sulfate anion becomes tied up in the reaction which allegedly forms a ferrous sulfate-glycine complex. Whether the sulfate actually participates in the reaction or is merely present as the salt of an alkali metal, it nevertheless is present in the reaction mixture. Thus, in many cases, the sulfate interferes with the total reaction and art absorption of the chelate. Such products are difficult to purify. While sodium sulfate, per se, is water soluble, the reaction between a metal sulfate and an amino acid is never carried to 100% completion and the sulfate ion is always present. The same holds true for the presence of chloride ions when utilizing a metal chloride salt for amino acid chelate preparation.
Even if one were to attempt to wash out the excess sulfate or chloride ions with repeated washes, such an attempt would likely be counter productive inasmuch as glycine and other amino acid ligands are also soluble to a degree. Hence, depending upon pH, the unreacted ligands or weakly held ligands could also be removed along with the unwanted anions.
As mentioned, in order to manufacture amino acid chelates, it generally requires that the metal salt and the ligand both be dissolved in water. One problem with this is employing metal salts that are soluble but essentially free from anions that can interfere with the chelation process. This is the subject of U.S. Pat. Nos. 4,599,152 and 4,830,716, both of which are incorporated by reference.
In the past, if certain soluble metal salts, such as sulfates, were used as a mineral source for chelation purposes, the resulting anions interfered with the chelation process. For example, the attraction between the lone pair of electrons on the amine group of an amino acid and a hydrogen ion is strong. This is why glycine is represented by the zwitterionic structure +H3NCH2COOxe2x88x92. This strong attraction for the hydrogen ion explains why amino acids are weak acids, e.g., the glycine is not easily deprotonated. In water, only about 0.5% of the glycine typically disassociates and releases a hydrogen ion. Additionally, in the prior art, the introduction of mineral acid salts into solution, such as copper sulfate, resulted in the creation of copper ions which compete with the hydrogen ion for the lone pair of electrons on the NH2group. Unfortunately, the equilibrium favors the majority of the amino groups remaining protonated. Thus, in order to efficiently chelate metal ions from certain soluble salts, it becomes desirable to render the interfering ions inactive or use soluble metal salts with non-interfering ions, such as oxides or hydroxides.
The present invention comprises compositions and methods of manufacturing electrically neutral amino acid chelates free of interfering ions. These amino acid chelates are prepared by reacting an calcium oxide and/or hydroxide, an amino acid, and a soluble metal sulfate salt in an aqueous solution at a ratio sufficient to allow substantially all of the ions present in solution to react. Thus, a metal amino acid chelate, calcium sulfate, and water are formed without the presence of any significant amounts of interfering ions. The metal amino acid chelates produced will have a ligand to metal molar ratio from about 2:1 to 3:1, depending on the valency of the metal, e.g., Fe(II) forms 2:1 and Fe(III) forms 3:1.
Before the present invention is disclosed and described, it is to be understood that this invention is not limited to the particular process steps and materials disclosed herein because such process steps and materials may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only. The terms are not intended to be limiting because the scope of the present invention is intended to be limited only by the appended claims and equivalents thereof.
It must be noted that, as used in this specification and the appended claims, the singular forms xe2x80x9ca,xe2x80x9d xe2x80x9can,xe2x80x9d and xe2x80x9cthexe2x80x9d include plural referents unless the content clearly dictates otherwise.
xe2x80x9cElectrically neutralxe2x80x9d refers to the final product of the reaction in which essentially all of the reactants have formed product such that there is no net charge, i.e., the amino acid chelate in particular is uncharged.
xe2x80x9cInterfering ionxe2x80x9d is meant to include any cation or anion which would hinder the formation of the amino acid chelate and which remains in the composition as a charged ion that has not reacted to form an amino acid chelate, calcium sulfate, or water.
xe2x80x9cMetal amino acid chelatexe2x80x9d or xe2x80x9camino acid chelatexe2x80x9d shall include metal ions bonded to amino acid ligands forming heterocyclic rings. The bonds may be coordinate covalent, covalent and/or ionic at the carboxyl oxygen group. However, at the xcex1-amino group, the bond is typically a coordinate covalent bond. Preferred amino acids include all of the naturally occurring amino acids.
xe2x80x9cMetalxe2x80x9d is meant to cover all nutritionally relevant metals that are more soluble as sulfate salts that calcium sulfate. Though calcium is a metal, for purposes of the present disclosure, calcium is excluded within this definition unless the context clearly dictates otherwise.
xe2x80x9cSoluble metal sulfatexe2x80x9d or xe2x80x9csoluble metal sulfate saltxe2x80x9d include all divalent or trivalent metals that are more soluble than calcium sulfate when in the form of a sulfate salt. Preferred soluble metal sulfate salts are comprised of at least one nutritionally relevant metal.
xe2x80x9cNutritionally relevant metalsxe2x80x9d include metals that are known to be needed by living organisms, particularly plants and mammals, including humans. Metals such as copper (Cu), zinc (Zn), iron (Fe), cobalt (Co), magnesium (Mg), manganese (Mn), and/or chromium (Cr), among others, are exemplary of nutritionally relevant metals.
Essentially, the present invention includes compositions and methods of manufacturing electrically neutral amino acid chelates free of interfering ions. These chelates are prepared by reacting 1) calcium oxide and/or hydroxide, 2) one or more amino acid, and 3) a soluble metal sulfate salt in an aqueous solution at a ratio sufficient to allow substantially all of the ions present in solution to react forming a metal amino acid chelate, calcium sulfate, and water, and wherein the metal amino acid chelate has a ligand to metal molar ratio from about 2:1 to 3:1.
The compositions and methods disclosed herein are similar to the invention described in the patent application filed of even date herewith and having U.S. Ser. No. 09/686,046 filed Oct. 11, 2000 which is incorporated herein by reference. A major difference between the present invention and that disclosed in the U.S. Ser. No. 09/686,046 application is that in the present invention, the amino acid chelates themselves are electrically neutral (not charged) and have a ligand to metal molar ratio from about 2:1 to 3:1. The U.S. Ser. No. 09/686,046 application discloses compositions and methods of preparing amino acid chelates that are charged having a hydroxide counter ion, and which have a ligand to metal molar ratio from about 1:1 to 2:1.
The amino acid to be used in the present invention is preferably one or more of the naturally occurring amino acid selected from the group consisting of alanine, arginine, asparagine, aspartic acid, cysteine, cystine, glutamine, glutamic acid, glycine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, ornithine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, and combinations thereof. However, dipeptides, tripeptides, and tetrapeptides formed by any combination of the naturally occurring amino acids can also be used. The metal should be more soluble as a sulfate salt than calcium sulfate. Exemplary metals include those selected from the group consisting of Cu, Zn, Fe, Cr, Co, Mg, Mn, and combinations thereof. Therefore, the metal reactant is preferably provided as a sulfate salt selected from the group consisting of copper sulfate (CUSO4), zinc sulfate (ZnSO4), ferrous sulfate (FeSO4), manganese sulfate (MnSO4), cobalt sulfate (COSO4), magnesium sulfate (MgSO4), ferric sulfate [Fe2(SO4)3], chromic sulfate [Cr2(SO4)3], and combinations thereof.
While not wanting to be bound by any theory, possible mechanism describing the reaction by which the amino acid chelates of the present invention are prepared may be broken down into two steps. Step A involves the reaction of one or more amino acids with a soluble calcium oxide and/or hydroxide in an aqueous solution forming a calcium amino acid chelate or complex product. Step B involves the reaction of a soluble metal sulfate salt with the calcium amino acid chelate or complex product formed in Step A. The calcium is displaced by the metal forming a metal amino acid chelate having a ligand to metal molar ratio from 2:1 to 3:1. Further, the calcium reacts with the sulfate anion to form an inert and highly insoluble calcium sulfate precipitate.
The reactions used to prepare electrically neutral amino acid chelates essentially free of interfering ions and having a ligand to metal molar ratio from about 2:1 to 3:1 are shown below. Formulas 3a and 3b illustrate the production of an amino acid chelate having a 2:1 ligand to metal molar ratio: 
In Formulas 3a and 3b above and 4a and 4b below, H(AA) is an amino acid selected from the group consisting of naturally occurring amino acids and combinations thereof. H, when disassociated from AA, is a hydrogen ion donor from the carboxyl group present on the amino acid. M is a nutritionally relevant metal having a valency of +2 (excluding calcium) such as Cu, Zn, Fe, Co, Mg, and/or Mn.
To illustrate this mechanism further, consider the reactants copper sulfate, calcium oxide, and glycine. First, one mole of calcium oxide is reacted with two moles of glycine. After allowing the calcium and glycine to react, copper sulfate is added. The log of the equilibrium constant at zero ionic strength for the reaction Ca2++(Gly)xe2x88x92 less than ⇄ greater than Ca(Gly)+, where (Gly)xe2x88x92 represents a glycine anion, is 1.39. By comparison, for the same reaction with copper rather than calcium, the log of the equilibrium constant is 8.56. Though a literature value is not given for the equilibrium constant for the attachment of two glycinate anions to a calcium cation, the principle that copper has stronger affinity for glycine ligands than does calcium is demonstrated by these values.
It is important to note that the reactants, i.e., CaO, H(AA), and MSO4, may be added in any order. For example, all three reactants may be added simultaneously or the amino acid and the soluble metal salt may be added before the calcium oxide or hydroxide. However, it is important to note that the above equation must be balanced so that the final products, M(AA)2, CaSO4, and water, are free of interfering ions and is electrically neutral. Therefore, notwithstanding Formula 3a and 3b which illustrates a preferred mechanism, the general formula of the present invention when a 2:1 ligand to metal molar ratio is desired may be represented by Formulas 4a and 4b as follows: 
For purposes of the present invention, multiple metals, amino acids, salts, etc., may be used in the context of the present invention as well. However, metals having a proper valency state should be used, e.g., Cu, Zn, Fe(II), Co, Mg, Al and/or Mn for amino acid chelates having a ligand to metal molar ratio of about 2:1.
General Formula 4 above may be modified to prepare amino acid chelates having a ligand to metal molar ratio of about 3:1 using metals having a valency of +3, e.g., Fe(III), Cr, etc. The general formula of the present invention when a 3:1 ligand to metal molar ratio is desired may be represented by Formulas 5a, and 5b as follows: 
In Formulas 5a and 5b, H(AA) is an amino acid selected from the group consisting of naturally occurring amino acids and combinations thereof. H, when disassociated from AA, is a hydrogen ion donor from the carboxyl group present on the amino acid. Mxe2x80x2 is a nutritionally relevant metal having a valence of +3 such as Fe(III) and/or Cr.
It is important to note that though the compositions and methods of the present invention provide electrically neutral amino acid chelates free of interfering ions, calcium sulfate is always a byproduct. Therefore, the calcium sulfate may be substantially separated out of the compound by methods commonly known in the art while the chelate is still in solution. Alternatively, the calcium sulfate may remain in the compound as a stabilizer or for other purposes.
To cite a specific example of a pure and electrically neutral amino acid chelate having a ligand to metal molar ratio of about 2:1, the following reactions steps may be followed. First, one mole of calcium oxide (CaO) may be reacted with two moles of glycine to form a reaction product of calcium bisglycinate in water. The problems associated with a hydrogen ion being bound to the amine group are resolved because it has reacted with the oxygen from the CaO to form water. To this solution, copper sulfate (CUSO4) is added. This allows for the disassociated copper ions to react with the calcium bisglycinate as the strength of bonding between a metal and a glycinate anion is stronger than the calcium glycinate bond. As such, the calcium is displaced by the copper forming copper bisglycinate. The calcium ions that are displaced in turn react with the sulfate anions (from the CuSO4) forming calcium sulfate which is sparingly soluble and essentially inert.
The present invention also encompasses drying of the chelate solution when appropriate to provide a powder for some uses, e.g., human, animal, and plant nutrition. However, with some applications, it may be desired that the chelate remain in solution, e.g., foliar use. If drying the chelate to form a particulate, any conventional drying technique as is known in the art may be used. For example, if spray drying, bulk density of the powder produced in a spray dryer is affected by the mesh size of the nozzles in the dryer, the pump pressure, and the percent of total solids in the solution to be dried. In general, the higher the total solids, the greater the bulk density of the resulting powder. A greater bulk density also reduces the electrostatic properties of the spray dried powder. For example, the presence of the calcium sulfate (terra alba) suspended in the metal amino acid chelate solution by continual agitation will increase the total solids to be dried, thus, increasing the ultimate bulk density of the dried chelate.
The increased bulk density of the dried product may have at least three distinct advantages. First, the dried product is less hygroscopic due to the increased density and due to the fact that calcium sulfate salt is less hygroscopic than the amino acid chelate, which through the drying process have waters of hydration removed. Second, a more dense particle is less electrostatic. This potentially reduces the cleanup time in a mixer when the chelate is blended with other food or pharmaceutical substances and enhances mixing characteristics when powdered chelates are blended with these other substances. Third, when the amino acid chelate is mixed with calcium sulfate, the presence of calcium sulfate stabilizes the amino acid chelate in an acidic solution.